Tissue engineering aims to create functional biologic prostheses by suspending dissociated cells into a biodegradable polymer scaffold upon which new tissue forms. Better means are urgently needed to compare engineered tissues, particularly cartilage and other soft tissues, with normal tissue. Histology is slow, cumbersome, and destructive because it requires first sectioning the tissue, then staining it. In addition, the quantitative reliability of histology is marginal owing to high variability of the stain intensity and color. This Phase I SBIR application aims for a quantum leap forward in tissue assessment, which will be realized by combining a unique implementation of time-resolved fluorescence spectroscopy with Monte-Carlo code that models light propagation in tissue. Using articular cartilage constructs as a model system, our team will demonstrate quantitative tracking of collagen expression in the chondrocytes, chondrocyte proliferation, and the spatial distribution of the collagen cross-links forming adjacent to the chondrocytes in the extracellular matrix over the entire growth process. Specific Aim 1 is to track the activity of GFP that serves as a marker of collagen synthesis in chondrocytes and Specific Aim 2 is to track the activity of collagen cross-linker formation as it develops in the region around the chondrocytes. Once successfully demonstrated, these capabilities will make it possible to test a vastly expanded number of variables that potentially affect tissue growth and the ultimate function of the construct. Tests of high throughput tissue engineering in Phase II could ultimately lead to prostheses that perform better and for a longer period after implantation. The data and scientific knowledge gained during the Phase I effort also has potential applications in real-time medical diagnostics and wound healing. Tissue engineering creates functional biologic prostheses by growing cells that form new tissue on a biodegradable polymer scaffold. The goal of this Phase I SBIR application is improved techniques to test the laboratory grown tissues before they are implanted, resulting in prostheses that perform better and last longer. [unreadable] [unreadable] [unreadable]